Part:BBa_K4342025

recJ Integration Cassette

Introduction

The 2022 UT Austin iGEM Team’s Part Collection provides a number of DNA sequences and procedures for genetically engineering Acinetobacter baylyi ADP1. We were able to effectively engineer ADP1's genome using a two-step genetic engineering protocol. See the Engineering Page for more details on how we modified ADP1's genome. On this page, we explain how our part collection can be used alongside this two-step protocol to delete ADP1 genes, insert DNA sequences into any chromosomal location, and engineer an ADP1-based biosensor to detect any DNA sequence of interest. We hope this part collection guides future iGEM teams in engineering ADP1 and utilizing ADP1’s flexibility to tackle any challenge in synthetic biology.

Categorization

For our parts collection, we categorize our parts into the following categories:

Upstream - An Upstream basic part is a DNA sequence directly upstream of a target gene. These basic parts are homology flanks that are used for ADP1 Genetic Engineering. Examples include the ACIAD2049 Upstream for P. destructans detector (BBa_4342003) and pbpG Upstream (BBa_4342011).

Downstream - A Downstream basic part is a DNA sequence directly downstream of a target gene. These basic parts are homology flanks that are used for ADP1 Genetic Engineering. Examples include ACIAD2049 Downstream for P. destructans detector (BBa_4342004) and pbpG Downstream (BBa_4342012).

Integration Cassettes - An "Integration" cassette is a composite part consisting of an "Upstream" basic part, the tdk/kan basic part (BBa_4342000), and a "Downstream" basic part. These parts are designed to use in the first transformation step in ADP1 Genetic Engineering. Examples include the ACIAD2049 Integration cassette (BBa_4342019) and the acrB Integration cassette (BBa_4342023).

Rescue Cassettes - A "Rescue" cassette is a composite part consisting of an "Upstream" basic part, an optional genetic device, and a "Downstream" basic part. These parts are designed to use in the second transformation step in ADP1 Genetic Engineering. Examples include the ACIAD2049 Rescue cassette (BBa_4342020, Upstream + Downstream), the YFP Rescue cassette (BBa_4342030, Upstream + Genetic Device + Downstream), and the nptII Detector Rescue cassette (BBa_4342031, Upstream + Composite Part + Downstream).

Genetic Device - A "Genetic Device" is a basic part that can be any DNA sequence to be integrated into ADP1. Examples include the CymR YFP (BBa_4342008) and the nptII Broken Gene (BBa_4342015).

We further categorize each part with a standardized Golden Gate Assembly (GGA) Type 1-8 Overhang [2]. Each type is ligated to a complementary type (ex. Type 2 can be ligated to Type 1 and Type 3). Moreover, some parts contain consecutive GGA Type numbers, such as Type 234. These DNA sequences start with a Type 2 Overhang and end with a Type 4 Overhang (ex. tdk/kan cassette (BBa_4342000).

recJ Integration Cassette is categorized as a Type 1-5 Integration cassette in our part collection.

Usage and Biology

recJ is a gene in Acinetobacter baylyi ADP1 that codes for a single-stranded DNA exonuclease that degrades short DNA in the cytoplasm [3]. Knocking out this gene allows for the integration of other DNA sequences in its place, and can also improve transformation frequency for small donor DNA less than 500 bp in length [3]. recJ also provides an additional ADP1 chromosomal location where other DNA sequences can be integrated. Using this part, we demonstrate that recJ can be deleted from ADP1's genome.

Design

The recJ Integration part comprises the 3695 bp region combining the recJ Upstream (BBa_4342013), tdk/kan (BBa_4342000) cassette, and recJ Downstream (BBa_4342014) parts.

• Please note that BsaI restriction sites have been removed to meet RFC[1000] BioBrick Assembly Compatibility. To see in-depth primer design, please see Figure 4 on the Engineering Page for more details on how to design primers containing the correct GGA Type Overhang and restriction sites.

Step 1

The recJ Integration cassette is designed to allow for successful transformant selection on Kanamycin (Kan) via the kanR gene (Figure 1).

Fig. 1. First-Step Integration of the tdk/kan cassette in place of an ADP1 Target Gene (recJ).

Step 2

The tdk/kan cassette can subsequently be knocked out to create a 4 bp minimal scar deletion of recJ via BsmBI digestion. During this reaction, a recJ Rescue cassette (BBa_4342026) is constructed. We use the recJ Rescue cassette to select for successful transformants on Azidothymidine (AZT) (Figure 2).

Fig. 2. Second-Step Removal of the tdk/kan cassette.

Characterization

To confirm that we successfully created this part, we transformed the recJ Integration cassette into ADP1-ISx [3]. First, we ran gel electrophoresis on our unpurified GGA product, containing the recJ Integration cassette (Figure 3).

Fig. 3. Synthesis of recJ Integration Cassette. BsaI GGA of recJ Upstream and recJ Downstream parts with pBTK622, containing the tdk/kan cassette. Lane 1: 3.7 kb tdk/kan ligated to homology flanks, 3.3 kb pBTK622 plasmid, 1.7 kb tdk/kan cassette, 1 kb homology flanks. Lanes 2 (no T7 DNA Ligase) and 3 (no homology flanks) serve as controls.

After confirmation the part was present in the GGA reaction, we proceeded with first-step integration of the tdk/kan cassette and selected for successful transformants on LB-Kan plates (Figure 4). Then, we picked colonies to inoculate for second-step removal of the tdk/kan cassette.

Fig. 4. Successful Transformation of the tdk/kan Cassette. Growth on the +DNA LB-Kan plates shows that the tdk/kan cassette was inserted into the recJ genome location in ADP1. The -DNA LB-Kan plates serve as a negative control for our transformation.

References

[1] Suárez, G.A., Dugan, K.R., Renda, B.A., Leonard, S.P., Gangavarapu, L.S., and Barrick, J.E. (2020). Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining. Nucleic Acids Research 48, 4585–4600. 10.1093/nar/gkaa204.

[2] Lee, M.E., DeLoache, W.C., Cervantes, B., and Dueber, J.E. (2015). A highly characterized yeast toolkit for modular, multipart assembly. ACS synthetic biology 4, 975–986. 10.1021/sb500366v.

[3] Gomez, M. J., & Neyfakh, A. A. (2006). Genes involved in intrinsic antibiotic resistance of Acinetobacter baylyi. Antimicrobial agents and chemotherapy, 50(11), 3562-3567. https://doi.org/10.1128/AAC.00579-06

[4] Suárez, G. A., Renda, B. A., Dasgupta, A., & Barrick, J. E. (2017). Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx. Applied and environmental microbiology, 83(17), e01025-17. https://doi.org/10.1128/AEM.01025-17

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1720
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 436
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3326
  Illegal BamHI site found at 1714
  Illegal BamHI site found at 3554
  Illegal XhoI site found at 1876
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1720
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1720
  
• 1000
  COMPATIBLE WITH RFC[1000]